ABSTRACT Our research on polyketide assembly lines is helping bring about a paradigm shift for how sets of component enzymes cooperate to biosynthesize polyketide natural products. The updated definition of a module, with the ketosynthase domain positioned at its downstream end, affects every level of modular polyketide synthase enzymology. Each of these levels must be further explored to achieve our long-term goal of reprogramming polyketide assembly lines to synthesize designer molecules and accelerate the drug discovery process. Our highest-resolution proposal is to study how ketosynthases gatekeep such that only one type of polyketide intermediate is selected by a module to be further elongated by the downstream assembly line (Specific Aim 1). This will be accomplished through methodology we have developed to crystallographically observe polyketides bound in ketosynthase active sites and measure the activity of ketosynthases mutated at suspected gatekeeping residues; engineered triketide synthases will also aid in this effort. From structures determined by our lab and others, we hypothesize that several uncharacterized domain interfaces are present within modules. We seek to structurally elucidate these interfaces within the context of the newly-defined module (Specific Aim 2). Thus, through crystallography and cryo-electron microscopy the structures of multidomain fragments possessing upstream processing enzymes and a downstream ketosynthase will be determined. We will also continue our efforts to characterize transient interfaces that form during the reaction cycle as acyl carrier protein domains present polyketide intermediates to cognate enzymes for catalysis. Our lab has collected several pieces of structural evidence for higher-order architecture. In the bacillaene polyketide synthase, a three-helix element adjacent to the ketosynthase domain seems to zipper homodimeric assembly lines into ~100 MDa assembly sheets observable within Bacillus subtilis cells. We propose to understand the structures of such biosynthetic megacomplexes by reconstituting them in vitro and observing them through electron microscopy (Specific Aim 3). Our lab is already visualizing Pks12 from Mycobacterium tuberculosis both in its ?bimodular? and its polymeric assembly line states. We seek to determine how modules stack to construct higher-order architecture in such systems. Through investigations at each of these levels, an overall picture of the architectures and activities of polyketide assembly lines will emerge that will be particularly significant to the future engineering of these medicinally-relevant molecular machines.